Versions of Cas9 derived from three different species have been exploited to edit genes in human cells. These Cas9 proteins recognize different PAM sequences. Cas9 originated from Streptococcus pyogenes (SpCas9) recognizes 5′-NGG-3′ PAM sequences and, to a lesser extent, 5′-NAG-3′. Cas9 from Streptococcus thermophilus (StCas9) (Cong et al., 2013 (link)) and that from Neisseria meningitidis (NmCas9) (Hou et al., 2013 (link)) recognizes 5′-NNAGAAW-3′ (W = A or T) and 5′-NNNNGMTT-3′ (M = A or C), respectively. The degeneracy in PAM recognition by Cas9 must be accounted for when searching for potential off-target sites. In the case of SpCas9, Cas-OFFinder first compiles all the 23-bp DNA sequences composed of 20-bp sequences corresponding to the sgRNA sequence of interest and the 5′-NRG-3′ PAM sequences (Fig. 1A). Cas-OFFinder then compares all the compiled sequences with the query sequence and counts the number of mismatched bases in the 20-bp sgRNA sequence.

(A) The scheme of Cas-OFFinder. (B) The workflow of Cas-OFFinder. (C) Running time per target site as a function of the number of input target sites via CPU (black squares) and GPU (red circles)